Capillary electrophoresis is a technique of considerable interest in the analysis of biological mixtures, particularly mixtures of small peptides, proteins and nucleic acids, since it can be used on extremely small samples and permits the use of high voltages which produce separations at high speed. Capillaries also offer the advantage of permitting on-line spectroscopic detection, a simple and highly efficient means of detection which avoids dilution of the solutes as well as any inaccuracies due to peak broadening or mixing upon emergence of the solutes from the capillary. Capillary electrophoresis in general encompasses various types of electrophoretic separations, most notably free zone electrophoresis and isoelectric focusing. Free zone electrophoresis is performed with a buffer solution as the separation medium. Solute separation and detection are performed in a single step as the solutes travel continuously at rates varying with their charge-to-mass ratio. The electric current thus serves both to separate the solutes into zones and to draw the zones in sequence across a detection point. Isoelectric focusing is performed in a medium containing a mixture of ampholytes with a pH gradient extending along the length of the capillary. In a focusing stage, the electric current causes each solute to travel until it reaches the location at which the pH is equal to the isoelectric point of the solute. The focusing stage is complete when all solutes are stabilized at their respective positions along the capillary, and this is followed by a mobilization stage in which the entire solute pattern is mobilized by any of various techniques toward or past a detection point.
A phenomenon which frequently occurs in capillary electrophoresis, particularly when the capillary is made of a silica-containing material, is electroendosmosis, also referred to as electroosmotic flow. Electroendosmosis arises from an electrokinetic potential existing between the wall of the solid element and the liquid separation medium adjacent to the wall. The flow which is caused by this potential is a bulk flow which occurs when an electric field tangential to the solid surface is imposed on the separation medium.
Bulk flow due to electroendosmosis has a potentially major effect in capillaries. Such bulk flow impairs the separation since it tends to cause mobilization of all solutes regardless of their electrophoretic mobility by mobilizing the separation medium itself, thereby interfering with the differentials in mobilization attributable to individual solute response. In extreme cases, this causes peak broadening and loss of resolution. Also, the electroendosmotic force varies from one run to the next as proteins become nonspecifically absorbed and desorbed at the capillary surface. This detracts from the reproducibility of the separation.
Electroendosmosis is commonly suppressed in capillaries by a coating on the interior capillary wall. The coating generally consists of neutral groups covalently bound to the capillary surface, eliminating any charged groups which are exposed and shielding the liquid medium adjacent to the wall from any charged groups remaining which are near the surface. In some cases, however, these coatings tend to deteriorate with extended use or with exposure to harsh solutes or separation media. This limits the useful life of capillaries and, when capillaries with partially deteriorated coatings are used in isoelectric focusing, the deterioration limits the length of the focusing time for any single run.